The present invention is directed to apparatus for use in diagnostic molecular pathology and, more particularly, to such an apparatus used for the automated staining and/or treating of tissue samples mounted on microscope slides.
Molecular pathology is the examination at a molecular level of the DNA, mRNA, and proteins that cause or are otherwise associated with disease. From this examination important information about patient diagnosis, prognosis, and treatment options can be elucidated. The practice of molecular pathology is generally divided into two main areas: (i) analysis of DNA, mRNA, and proteins in intact cells (in-situ), and (ii) analysis of these biological materials after they have been extracted from tissues. The first category, to which the present invention is directed, has the advantage that it allows the pathologist or scientist to study the histopathologic architecture or morphology of the tissue specimen under the microscope at the same time that the nucleic acid or proteins are being assayed. These techniques include immunohistochemistry (IHC) which looks at proteins, in-situ hybridization (ISH) which looks at nucleic acids, histochemistry (HC) which looks at carbohydrates, and enzyme histochemistry (EHC) which looks at enzyme chemistry. For example, ISH can be used to look for the presence of a genetic abnormality or condition such as amplification of cancer causing genes specifically in cells that, when viewed under a microscope, morphologically appear to be malignant. ISH is also useful in the diagnosis of infectious diseases as it allows detection not only of a microbial sequence but also of precisely which cells are infected. This may have important clinicopathologic implications and is an effective means to rule out the possibility that positive hybridization signal may have come from an adjacent tissue of no clinical concern or from blood or outside contamination.
IHC utilizes antibodies which bind specifically with unique epitopes present only in certain types of diseased cellular tissue. IHC requires a series of treatment steps conducted on a tissue section or cells (e.g. blood or bone marrow) mounted on a glass slide to highlight by selective staining certain morphological indicators of disease states. Typical steps include pretreatment of the tissue section to remove the paraffin and reduce non-specific binding, retrieval of antigens masked by cross-linking of the proteins from the chemical fixatives, antibody treatment and incubation, enzyme labeled secondary antibody treatment and incubation, substrate reaction with the enzyme to produce a fluorophore or chromophore highlighting areas of the tissue section having epitopes binding with the antibody, counterstaining, and the like. Most of these steps are separated by multiple rinse steps to remove unreacted residual reagent from the prior step. Incubations can be conducted at elevated temperatures, usually around 37xc2x0 C., and the tissue must be continuously protected from dehydration. ISH analysis, which relies upon the specific binding affinity of probes with unique or repetitive nucleotide sequences from the cells of tissue samples or bodily fluids, requires a similar series of process steps with many different reagents and is further complicated by varying temperature requirements.
In view of the large number of repetitive treatment steps needed for both IHC and ISH, automated systems have been introduced to reduce human labor and the costs and error rate associated therewith, and to introduce uniformity. Examples of automated systems that have been successfully employed include the NEXES(copyright) and Gen II(copyright) staining Systems available from Ventana Medical Systems (Tucson, Ariz.) as well as the system disclosed in U.S. Pat. No. 5,654,199 to Copeland et al. These systems employ a microprocessor controlled system including a revolving carousel supporting radially positioned slides. A stepper motor rotates the carousel placing each slide under one of a series of reagent dispensers positioned above the slides. Bar codes on the slides and reagent dispensers permits the computer controlled positioning of the dispensers and slides so that different reagent treatments can be performed for each of the various tissue samples by appropriate programming of the computer.
The aforementioned staining systems include either a hot air blower or a heat lamp to warm the samples above laboratory ambient temperatures for steps requiring elevated temperatures. Heating the slide improves staining quality by acceleration of the chemical reaction and can permit a reaction temperature more closely matching body temperature (about 37xc2x0 C.) at which antibodies are designed to react. While such convection or radiant heating systems have been generally suitable for IHC, which is antibody based, they are less suitable for ISH, which is nucleic acid based and requires higher and more precise temperature control. In order to denature the DNA double helix of both the target sample and the probe so as to render them single stranded, the temperature must be raised above the melting point of the duplex, usually about 94xc2x0 C. At the same time it is imperative that the sample not be overheated past 100xc2x0 C. as doing so destroys cell morphology making it difficult to view under a microscope. Precise temperature control is also required in ISH to effect probe hybridization at the desired stringency. The selected temperature must be low enough to enable hybridization between probe and template, but high enough to prevent mismatched hybrids from forming. It would be desirable, therefore, to have an automatic tissue staining apparatus which can control the temperature of reactions with enough precision for most ISH applications.
In addition, even if heaters are used in combination with the slide holders, the heating of the slides may be inaccurate. For example, U.S. Pat. No. 5,645,114 to Bogen et al. discloses a dispensing assembly adapted to carry a plurality of microscope slides. Individual slide holders containing resistive heating units are provided. However, with the assembly taught by Bogen et al., the actual temperature of the slide placed on top of the heater may be different than the temperature set for the heater. This is due to the fact that different heaters have different heating profiles (i.e., there may be variations in temperature on the platform of the heater). This is also due to the fact that the temperature sensor, which determines whether the heater is heating to the correct temperature, is not placed in the same position as that of the slide. Instead, the temperature sensor is typically placed in an area away from the slide, such as on the underside of the heater, thereby potentially resulting in an inaccurate heating of the slides. It would be desirable, therefore, to have an automatic tissue staining apparatus wherein a slide can be accurately heating to a specific predetermined temperature.
The present invention is directed to apparatus and methods for automatically staining or treating multiple tissue samples mounted on microscope slides so that each sample can receive an individualized staining or treatment protocol even when such protocols require heating. More specifically, the apparatus is a computer controlled, bar code driven, staining instrument that automatically applies chemical and biological reagents to tissue or cells mounted or affixed to standard glass microscope slides. Up to 20 slides are mounted in a circular array to a carousel which rotates, as directed by the computer, placing each slide under one of a series of reagent dispensers positioned above the slides. Each slide receives the selected reagents (e.g. DNA probe) and is washed, mixed and/or heated in an optimum sequence and for the required period of time. Tissue sections so stained or treated are then removed from the apparatus by the user to be viewed under a microscope by a medical practitioner who reads the slide for purposes of patient diagnosis, prognosis, or treatment selection. The computer controlled automation permits use of the apparatus in a xe2x80x9cwalk-awayxe2x80x9d manner, i.e. with little manual labor.
Slide temperature control is accomplished by a heating system according to the present invention. The heating system, in one embodiment, has a conversion buffer in between the desired temperature generator and the heating controller. The heating controller is in communication with a slide temperature sensor, which provides feedback to the heating controller, and a heater. Because the slide temperature sensor may inaccurately reflect the actual temperature on the slide, the heating controller may inaccurately control the heater and heat the slide to an undesired temperature. Thus, the conversion buffer compensates for potential inaccuracies of the heating controller. The desired temperature generator sends the xe2x80x9cdesired temperaturexe2x80x9d for the slide to the conversion buffer. The conversion buffer converts the xe2x80x9cdesired temperaturexe2x80x9d to an xe2x80x9cadjusted temperaturexe2x80x9d and sends it to the heating controller. The heating controller then controls the heater based upon the xe2x80x9cadjusted temperaturexe2x80x9d and the heating sensor. The conversion buffer may be generated during calibration of the machine. Similarly, the temperature which is sensed from the slide temperature sensor may be adjusted to reflect the actual temperature for the slide.
A key advantage of the present invention is that the slides can be accurately heated to a specific predetermined temperature.
Another advantage of the present invention is that it allows the calibration of the slide heaters so that during operation of the heaters, the slide is heated to the proper temperature.